Lymphoproliferative disorders in autoimmune diseases in Japan: Analysis of clinicopathological features and Epstein-Barr virus infection

Abstract

Lymphoproliferative disorders (LPD) occasionally develop in individuals with immune deficiencies such as immunosuppressive conditions and autoimmune diseases (AID). In our study, the clinicopathologic features and virus status were analyzed in 53 cases with LPD developing in rheumatoid arthritis (RA) and other AID. AID in only 4 of 53 patients had been treated with some sort of immunosuppressive therapy, including methotrexate. Median age at the diagnosis of LPD in AID was 60 years old with marked female predominance (M/F = 0.4). The median interval between the onset of AID and LPD development was 45 months, and longer in RA patients than in other AID (p < 0.01). The primary site of lymphoma was nodal in 21 cases and extra-nodal in 24, with clinical Stage I in 17, II in 5, III in 13, and IV in 13. Immunohistochemistry showed that 39 cases were B cell type, 10 were T cell type and 4 were Hodgkin lymphoma (HL). Then majority of B cell cases were diffuse large B cell lymphomas, and 2 were diffuse polymorphic type. EBER-1 in situ hybridization for Epstein-Barr virus (EBV) showed positive signals in tumor cells in 16 of 53 (30.2%) cases. The EBV-positive rate in T cell LPD (70%) was much higher than that in B cell LPD (12.8%) (p < 0.01). All 4 cases of HL were EBV-positive. Immunohistochemistry showed a latency II pattern of EBV infection (LMP-1⁺ and EBNA-2⁻). Five-year overall survival rate was 33%. Multivariate analysis showed that only type of AID was an independent factor for survival of patients, i.e., LPD in RA showed the most favorable prognosis. In conclusion, LPD in AID generally shared common features with sporadic LPD except for a much higher EBV-positive rate in T cell LPD. © 2003 Wiley-Liss, Inc.

Lymphoproliferative disorders (LPD) occasionally develop in individuals with immune deficiencies such as immunosuppressive conditions and autoimmune diseases (AID). Information has accumulated on LPD in immunosuppressive conditions. In the recent World Health Organization (WHO) classification, prior immunosuppressive diseases to the LPD are categorized into primary immune disorders, human immunodeficiency virus (HIV) infection, iatrogenic immunosuppression in patients receiving solid organ or bone marrow allografts, and iatrogenic immunosuppression associated with methotrexate (MTX).1

Individuals affected by AID, such as rheumatoid arthritis (RA) and dermatomyositis (DM), develop LPD at a frequency of 2.0–5.5 times higher than in the general population.2, 3, 4 These LPD were primarily B cell in nature and those with T cell phenotype comprise approximately 5% of cases in Western countries.1 Information on LPD developing in other kinds of AID such as systemic lupus erythematosus (SLE), progressively systemic scleroderma (PSS) and autoimmune hemophilic anemia (AIHA), however, is limited.

Methotrexate (MTX) is administered in patients with AID, especially RA, to suppress the hyperimmune state. This in turn might induce immunosuppression and provide a basis for the development of LPD.5, 6 This disease occasionally shows a polymorphous pattern of proliferation, Epstein-Barr virus (EBV)-association, and complete regression after withdrawal of MTX.7 Post-transplantation LPD (PT-LPD) and LPD in AID seems to share a common feature of employment of immunosuppressive therapy and development of LPD. In addition, an increased risk of LPD in patients with RA was reported even in the absence of MTX therapy.8 Hence, LPD in patients with AID might include a heterogeneous background of lymphoid proliferations.

In our study, the clinicopathologic features and virus conditions in 53 cases of LPD developing in AID were analyzed, and the results were compared to those in LPD with different backgrounds of immunosuppression with a review of the pertinent literature.

AID, autoimmune diseases; AIHA, autoimmune hemophilic anemia; ATL, adult T - cell leukemia/lymphoma; Cox-2, cyclooxygenase-2; CTL, cytotoxic T-lymphocytes; DM, dermatomyositis; EBNA, EB nuclear antigen; EBV, Epstein-Barr virus; HHV8, human herpes virus type 8; HIV, human immunodeficiency virus; HL, Hodgkin lymphoma; HTLV, human T-lymphotropic virus; LMP, latent membrane protein; LPD, Lymphoproliferative disorders; MTX, methotrexate; NSAID, non-steroidal anti-inflammatory drugs; PSS, progressively systemic scleroderma; PT-LPD, post-transplantation LPD; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; WHO, World Health Organization.

PATIENTS AND METHODS

Fifty-three patients with AID in whom LPD developed were selected for the current study: 13 through the review of Japanese journals, 31 through the “Annual of Pathological Autopsy Cases in Japan (1978–1997)” and 9 through consultation case files in the Department of Pathology, Osaka University. They were admitted to the hospitals during the period from 1997–2002. Histological specimens obtained by biopsy (36 cases) or autopsy (17 cases) were fixed in 10% formalin and routinely processed for paraffin-embedding. Histologic sections cut at 4 μm were stained with H&E and an immunoperoxidase procedure. All of the histologic sections were reviewed by 2 of the authors (Y.H., K.A.), and classified according to the WHO Classification.1 Adequate clinical information was available in all cases.

Immunohistochemical staining

Immunoperoxidase procedures (ABC method) were carried out in all cases as described previously.9 Monoclonal antibodies used in the present study, their suppliers, reactivities, and dilutions are shown in Table I. Before incubation, sections were pretreated with 1% trypsin in Tris buffer (pH7.8) for CD3ϵ, Ber-H2 and latent membrane protein (LMP)-1, by microwaving for 5 min in citrate buffer (pH 6.0) for CD8, CD16 and CD56, by microwaving for 5 min in 1 mM EDTA (pH8.0) for CD4, and by autoclaving at 121°C for 15 min in citrate buffer (pH 6.0) for EB nuclear antigen (EBNA)-2. As positive controls, a case of Hodgkin lymphoma (HL) and PT-LPD with proven positive immunoreactivities for LMP-1 and EBNA-2, respectively, were stained in parallel with the cases.

Table I. Antibody Panel

Antibody	Dilution	Reactivity	Supplier
L-26 (CD20)	1:200	B-lymphocytes	Kyowa Medex, Tokyo, Japan
CD3ε	1:100	T-lymphocytes	Dakopatts, Glostrup, Denmark
UCHL-1(CD45RO)	1:100	T-lymphocytes	Dakopatts, Glostrup, Denmark
N-CAM (CD56)	1:40	NK/T-lymphocytes	Zymed Laboratories Inc. San Francisco, CA
2H7 (CD16)	1:200	NK/T-lymphocytes	Novocastra, Newcastle, UK
TIA-1	1:500	Cytotoxic granules	Coulter, Hialeah, FL
1F6 (CD4)	1:200	Helper/inducer T-lymphocytes	Novocastra
C8/144B (CD8)	1:200	Suppressor/cytotoxic T-lymphocytes	Dakopatts
Ber-H2 (CD30)	1:10	Reed-Sternberg cells	Dakopatts
β F-1	1:50	TCR β receptor chain	Endogen, Woburn, MA
CSI-4 (LMP-1)	1:20	Latent membrane protein	Dakopatts
PE-2 (EBNA 2)	1:10	Epstein-Barr virus nuclear antigen 2	Dakopatts

In situ hybridization

EBV RNA-in situ hybridization (ISH) was carried out as described previously.10 As a positive control, the Raji cell line was used. As negative controls, the hybridizing mixture was employed with (i) sense probe and (ii) antisense probe after RNase (Sigma, St. Louis, MO) treatment.

DNA extraction

DNA was extracted from paraffin-embedded sections using chelating resin (Sigma). Briefly, 5 pieces of 5-μm section were cut from paraffin blocks, then suspended in 40% resin, boiled for 10 min and centrifuged for 5 min at 18,000g. Supernatant was used for each PCR analysis (1–10 ml). Preservation of DNA was confirmed by PCR amplification with primers specific for a 205-bp segment of the β-globin gene (Table II). Thirty-two cases of 36 LPD with B cell phenotype, 7 of 8 with T cell phenotype and 3 of 4 HL showed an amplified band of the expected size by β-globin PCR. These cases were used for the following analyses.

Table II. Oligonucleotide Primers Used For PCR

Gene	Sequences1	Direction
1 R = A/G, W = A/T, S = C/G, Y = C/T, M = A/C, N = A/C/G/T.
Beta-globin	5′-GGTTGGCCAATCTACTCCCAGG-3′	Upstream
	5′-CAACTTCATCCACGTTCACC-3′	Downstream
Epstein-Barr virus	5′-CCAGACAGCAGCCAATTGTC-3′	Upstream
	5′-GGTAGAAGACCCCCTCTTAC-3′	Downstream
Human herpes virus 8	5′-AGCCGAAAGGATTCCACCAT-3′	Upstream
	5′-TCCGTGTTGTCTACGTCCAG-3′	Downstream
Human T-lymphotrophic virus-1,2
tax region	5′-CGGATACCCAGTCTACGTGT-3′	Upstream
	5′-GAGCCGATAACGCGTCCATCG-3′	Downstream
pol region	5′-CTTCACAGTCTCTACTTGTGC-3′	Upstream
	5′-CGGCAGTTCTGTGACAGGG-3′	Downstream
Fr2A	5′-TGGRTCCGMCAGSCYYCNGG-3′
Fr3A	5′-ACACGGCYSTGTATTACTGT-3′
LJH	5′-TGAGGAGACGGTGACC-3′
VLJH	5′-GTGACCAGGGTNCCTTGGCCCCAG-3′

Detection of EBV, human herpes virus type 8 and human T-lymphotropic virus-1 genome

For detection of the EBV genome, 35 cycles of 94°C/58°C/72°C were carried out with primers designed to amplify a 129-bp segment in the BamHI-W fragment of the EBV genome.11 For the HHV-8 genome, 40 cycles of 94°C/60°C/72°C were used with primers for the 233-bp segment in the KS330 region.12 DNA extracted from Kaposi's sarcoma tissue containing human herpes virus type 8 (HHV-8) genome was used as positive control. For human T-lymphotropic virus-1 (HTLV-1) proviral DNA, 40 cycles of 94°C/58°C/72°C were used with tax primers for a 159-bp segment common to the HTLV-1 and HTLV-2 proviral genome13 and pol primers for a 119-bp segment specific to the HTLV-1 proviral genome.14

Analysis of immunoglobulin heavy chain gene

For rearrangement analysis of the immunoglobulin heavy chain gene, 2-step semi-nested PCR was carried out in 32 cases of LPD with B cell phenotype. The primers used in the semi-nested PCR were Fr2A, Fr3A, LJH and VLJH. The sequences of the primers and PCR conditions were the same as those described previously (Table II).15 Each PCR experiment included a sample without DNA template as a negative control and a sample with DNA extracted from a lymphoblastoid cell line as a positive control. The second PCR products were electrophoresed on 3% Metaphor (BioWhittaker Molecular Applications, Rockland, ME) agarose gel and stained with ethidium bromide to visualize the DNA under ultraviolet light.

Statistical analysis

Actuarial survival curves were calculated by the Kaplan and Meier method,16 and differences were examined by the log-rank test to detect significant prognostic factors.17 Factors examined were age, gender, type of AID, primary site of LPD lesion, stage of disease, type of histology, immunophenotype of proliferating cells, EBV positivity and type of medication for AID. Correlation between EBV positivity and primary site of tumor, immunophenotype of proliferating cells and histologic type was examined by the χ² test. The Mann-Whitney U-test was used to evaluate the difference in age at the LPD development between patients with and without EBV, and the interval from the onset of AID to LPD development between patients with or without MTX medication. Multivariate analysis was carried out with the Cox's proportional-hazards model to identify independent prognostic factors.18 A multiple comparison test (Scheffe's F-test)19 was used to evaluate the difference in age at the LPD development and duration between onset of AID and LPD development. A p-value of <0.05 was considered significant.

RESULTS

Clinical findings

The clinical features are summarized in Tables III and IV. All of the present patients had been suffering from AID until the development of LPD; RA in 22 cases, SLE in 10, DM in 9, PSS and AIHA in 7 each. Two of these patients suffered from SLE and AIHA. Two of the PSS patients suffered from Sjögren's disease, and one of RA patient suffered from Hashimoto's disease. Four patients with RA had been treated with low-dose MTX (2.5 mg/w for 72 months in 1 patient, 5.0 mg/w for 32 and 60 months in 2 patients, respectively, and 7.5 mg/w for 60 months followed by 36 mg/w for 11 months in 1 patient) and the remaining 49 patients were treated with non-MTX therapy, mostly including non-steroidal anti-inflammatory drugs (NSAID). Age of patients at the diagnosis of malignant lymphomas ranged from 23–83 (median 60) years. Male to female ratio was 15:38. The interval between the onset of AID and lymphoma development ranged from 1–312 (median 45) months in total and was significantly longer in patients with RA than in other AID (p < 0.01). Similarly, that in patients treated with MTX was significantly longer than those without MTX (p < 0.01). The primary site of LPD was nodal in 21 cases and extra-nodal in 24. The remaining 8 patients presented with advanced diseases, therefore the primary sites could not be determined. Based on the records of physical examinations, surgical notes, and pathological examination of the specimens, the Ann Arbor staging scheme was applied in all cases: Stage I in 17 patients, Stage II in 5 patients, Stage III in 13 patients and Stage IV in 13 patients.

Table III. Summary of Clinicopathological Findings in Lymphoproliferative Disorders Developing in Autoimmune Diseases1

	Total	RA	SLE2	DM	PSS	AIHA2
1 AID, autoimmune disease; UD, undetermined; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; DM, dermatomyositis; PSS, progressive systemic sclerosis; AIHA, autoimmune hemolytic anemis; MTX, methotrexate. 2 Two patients suffered with SLE and AIHA simultaneously. 3 p < 0.01 compared to RA.
Case number	53	22	10	9	7	7
Age (years)
Median	60	66	44	64	57	60
Range	23–83	23–76	25–83	35–80	56–65	28–83
Gender (male:female)	15:38	5:17	3:7	4:5	2:5	2:5
Duration between onset of AID and lymphoma (m)
Median	45	162	213	23	263	393
Range	1–312	24–312	8–228	1–51	3–144	2–84
Primary site
Nodal	21	10	1	4	5	1
Extranodal	24	11	7	3	1	3
UD	8	1	2	2	1	3
Clinical stage
I	17	6	6	3	1	1
II	5	3	0	2	0	0
III	13	7	1	2	5	0
IV	13	4	2	2	1	4
UD	5	2	1	0	0	2
Therapy for AID
MTX	4	4	0	0	0	0
Non-MTx	49	18	10	9	7	7
Outcome (%)
1 year-survival rate	52	84	383	153	03	433
5 year-survival rate	33	57	19	—	—	—

Table IV. Summary of Clinicopathological Findings in Lymphoproliferative Disorders Developing in Autoimmune Diseases1

	Total	B-cell				T-cell			HL
		Total	DLB	Pol	Others1	Total	PTL	Others2
1 DLB, Diffuse large B cell lymphoma; Pol, Polymorphic LPD; PTL, Peripheral T cell lymphoma, not specified; HL, Hodgkin lymphomas; AID, autoimmune disease; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; DM, dermatomyositis; PSS, progressive systemic sclerosis; AIHA, autoimmune hemolytic anemia. 2 Plasmacytoma 3 cases, follicular center cell lymphoma 1, mantle cell lymphoma 1, lymphoplasmacytic lymphoma 1. ^3Angioimmunoblastic T-cell lymphoma 1, anaplastic large cell lymphoma 2.
Case No.	53	39	31	2	6	10	7	3	4
Age (years)
Median	60	63	62	59	67	47	41	60	50
Range	23–83	28–80	28–80	58–60	57–75	23–83	23–83	58–65	41–57
Gender (male:female)	15:38	13:26	10:21	1:1	2:4	3:7	2:5	1:2	1:3
Kind of AID
RA	22	18	13	1	4	3	1	2	1
SLE	10	5	5	0	0	4	4	0	1
DM	9	7	6	0	1	2	2	0	0
PSS	7	5	4	0	1	0	0	0	2
AIHA	7	4	3	1	0	3	2	1	0
Primary site
Nodal	21	15	12	2	1	4	2	2	2
Extranodal	24	20	15	0	5	3	3	0	1
Undetermined	8	4	4	0	0	3	2	1	1
EBV
+ (%)	16 (30.2)	5 (12.8)	3 (9.7)	2 (100)	0 (0)	7 (70)	5 (71)	2 (67)	4 (100)
−	37	34	28	0	6	3	2	1	0
Prognosis (%)
1 year survival rate	52	60	56	50	80	29	17	67	33
5 year survival rate	33	41	42	—	27	—	—	—	—

Histological findings

Immunohistochemistry showed that 39 cases were B cell type, 10 were T cell type and 4 were HL (Fig. 1). The majority of B cell cases were diffuse large B cell lymphomas. Two cases showed polymorphous appearance consisting of the full-range of B cell maturation from immunoblasts to plasma cells, small- and medium-size lymphocytes and numerous cells with centrocytic and centroblastic appearance and were thus categorized as diffuse polymorphic type. Among T cell cases, peripheral T cell lymphoma, not specified, was the commonest, and there were no cases of ATL (Table V). Subtype of HL was mixed cellularity in 2 cases (1 = lymphocyte rich classical HL; 1 = lymphocyte depletion).

Figure 1. (a) Peripheral T cell lymphoma, not specified, developing in an 83-year-old male with SLE (Case 3 in Table V). There was polymorphous proliferation consisting of medium to large-sized tumor cells with irregular nuclei and small lymphocytes. The tumor cells were positive for a T cell marker (CD3ϵ), EBER-1 and LMP-1 protein. (a) H&E; (b) CD3ϵ; (c) EBER-1; (d) LMP-1. Scale bar = 25 μm.

Table V. Summary of LPD With T-Cell Phenotype and Hodgkin Lymphoma1

Case	His	Age/gender	Type of AID	Primary site	Stage	Treatment for lymphoma	Response	Follow-up (months)	EBV
1 AID, autoimmune disease; PTL, peripheral T cell lymphoma, not specified; AILD, angioimmnoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; HL, Hodgkin lymphoma; LR, lymphocyte rich; MC, mixed cellularity, LD, lymphocyte depletion; UD, undetermined; ND, not done; PD, progressive disease; DID, death due to intercurrent disease; A, alive; DT, death due to tumor.
1	PTL	23/F	RA	UD	IV	ND	No therapy	0 DID	+
2	PTL	30/M	SLE	Liver	I	Chemo + Rad	PR	20 DID	−
3	PTL	83/M	SLE	UD	IV	ND	No therapy	1 DID	+
4	PTL	25/F	SLE	Cervical LN	III	Chemotherapy	PD	2 DT	+
5	PTL	47/F	SLE	Intestine	UD	ND	No therapy	1 DT	+
6	PTL	75/F	DM	Skin	III	Chemotherapy	PD	3 DT	−
7	PTL	35/F	DM	Cervical LN	I	Chemo + Rad	PD	8 DT	+
8	AILD	65/F	RA	Cervical LN	II	Chemotherapy	PD	1 DT	+
9	ALCL	58/F	RA	Maxillar LN	III	Chemotherapy	CR	22 DT	+
10	ALCL	60/M	AIHA	UD	IV	Chemotherapy	PD	6A	−
11	HL, LR	57/M	PSS	Cervical LN	III	Chemotherapy	PD	2 DT	+
12	HL, MC	41/F	RA	Cervical LN	III	Chemotherapy	PR	36 DT	+
13	HL, MC	57/F	PSS	UD	III	ND	No therapy	0 DID	+
14	HL, LD	43/F	SLE	Stomach	I	ND	No therapy	7 DT	+

Immunoglobulin heavy chain gene analysis

Single or double bands of the expected size were detected in 30 of 32 cases with B cell LPD by at least one of Fr2A and Fr3A PCR, showing the monoclonal nature of the LPD cells (Fig. 2).

Figure 2. Immunoglobulin heavy chain gene rearrangement with Fr3A primer. Monoclonal single or double bands were observed in 30 of 32 samples examined. Remaining 2 samples (numbers 5 and 30) did not show the clonal band by the PCR analysis. Abbreviations: M, size marker; 1,2, diffuse polymorphic; 3–27, diffuse large B cell lymphoma; 28, follicular center cell lymphoma; 29, lymphoplasmacytic lymphoma; 30–32, plasmacytoma; N, negative control (water); P, positive control (Raji).

EBV analysis

ISH and PCR analyses were carried out to detect the EBV genome in the LPD samples. ISH showed positive signals in the nucleus of large cells with nuclear atypia in cases with NHL and the Reed-Sternberg cells in HL: 50–90% of the large cells and >90% of Reed-Sternberg cells exhibited positive signals. PCR showed 11 of 42 cases to be positive for EBV. Among them, ISH confirmed positive signals in the nucleus of proliferating cells in 9 cases but not in the remaining 2 cases. In contrast, ISH showed positive signals in 3 cases without positive products by PCR analysis and 4 cases that were not analyzed by PCR. These cases were considered as EBV-positive. As a result, 16 (30.2%) of 53 cases were regarded as EBV-positive LPD (Table IV). B cell lymphomas developing in all of 4 patients treated with MTX were EBV-negative. The EBV-positive rate in T cell lymphoma (70%) was much higher than that in B cell lymphoma (12.8%) (p < 0.01; χ² test). All 4 cases of HL were positive for EBV. Immunohistochemistry showed that tumor cells in all the EBV-positive cases expressed LMP-1 but not EBNA-2 protein. A positive control, PT-LPD, showed a positive immunoreactivity for EBNA-2. Age at the diagnosis of LPD was significantly younger in patients with EBV-positive LPD than in those with EBV-negative LPD(p < 0.05). The EBV-positive rate in nodal lesions (48%) was significantly higher than that in extra-nodal lesions (1.5%) (p < 0.01; χ² test).

HHV-8 and HTLV-1 analysis

PCR analyses for the HHV-8 genome were carried out in 37 cases with AID-LPD, giving negative results. PCR analyses for the HTLV-1 proviral genome were carried out in 10 cases with T cell phenotypes, giving negative results. Serum anti-HTLV-1 antibody was examined in 4 of 10 cases and also showed negative results.

Clinical outcome

Thirty-seven (69.8%) of the 53 patients received adjuvant therapy after the diagnosis of malignant lymphomas: chemotherapy in 28 patients, radiotherapy in 2 patients and combined chemo- and radiation therapy in 7 patients. The chemotherapeutic agents included cyclophosphamide, vincristine, doxorubicin, predonine, methotrexate, bleomycin, mitomycin C, mercaptopurine and melphalan. Reduction of immunosuppression and anti-CD20 antibody therapy were never employed. The treatment outcome was evaluated in 33 patients according to the guidelines of the International Workshop to standardize response criteria for NHL.20 Adequate information to determine the clinical outcome was not available in four patients. After chemo- or radiotherapy, 70% of 33 patients showed a complete or partial response. Three patients had no response to the therapy and 7 had progressive disease. Thirteen patients did not receive adjuvant therapy: 3 received surgical resection of the lesion and 10 did not receive any kind of therapies because LPD could not be diagnosed until the terminal stage or autopsy.

The follow-up period for survivors calculated from the date of diagnosis of LPD ranged from 1–144 (mean 20) months. The 1- and 5-year overall survival rates in all patients were 52.0% and 33.0%, respectively. Univariate analysis showed that the type of AID (p < 0.01), immunophenotype of proliferating cells (p < 0.05), histologic type of LPD (p < 0.01), and EBV positivity (p < 0.05) were significant prognostic factors, i.e., patients with RA, B cell phenotype, diffuse large B cell lymphoma, anaplastic large cell lymphoma, follicle center cell lymphoma or lymphoplasmacytic lymphoma and negativity for EBV showed a more favorable prognosis. Multivariate analysis showed that only the type of AID was a significant independent factor for overall survival (p < 0.01), i.e., RA patients showed a more favorable prognosis than patients with other AID.

DISCUSSION

Median age at the diagnosis of LPD in the present patients with AID was 60 years old, which was much higher than that in immunosuppressive diseases (36–40 years old)21, 22 and rather higher than that in immunocompetent individuals in Japan (54 years old).23 There was a marked female preponderance (M/F = 0.4), which was in contrast to male preponderance in immunocompetent (M/F = 1.7–1.8) and immunosuppressed patients (M/F is 5 in PT-LPD, 24 in AIDS-related lymphoma).21, 22, 23 This difference might reflect a difference in the gender ratio in the preceding immunodeficient conditions. As for the primary site of LPD, extranodal involvement predominated in AID patients, although it was not as prominent as observed in immunosuppressed patients.21, 22, 23 The interval between the onset of AID and the diagnosis of LPD was significantly longer in patients with RA than that in other AID. This longer interval in RA might be due to the use of NSAID containing cyclooxygenase-2 (Cox-2) inhibitor, thus suppressing the development of malignancies such as colon and stomach cancer.24, 25, 26

More than 80% of LPD in the present RA patients showed B cell immunophenotype, with diffuse large B cell lymphoma being most common (59%), whereas 40% and 29% of LPD cases in SLE and PSS were peripheral T cell lymphoma and HL, respectively. The distribution of T, B immunophenotype and HL in LPD in AID was similar to that in immunocompetent individuals in Japan.23, 27 NK/T cell lymphoma and ATL are relatively common in PT-LPD in Japan,21 but they are absent among LPD in AID. HTLV-1 infection, an oncogenic virus of ATL, might be due to transmission via blood transfusion during hemodialysis before renal transplantation in Japan. AID patients do not usually receive blood transfusion, and no cases of ATL were found in the current cases.

EBV DNA is detected in various kinds of malignant lymphomas. Irrespective of the causes, an immunodeficient state is considered to provide a basis for the development of malignant lymphomas, probably through the activation of EBV. The EBV-positive rate in PT-LPD is reported to be 63–95%,28 whereas that in sporadic LPD in Western country is 5%.29 Approximately 30% of the present cases were EBV-positive, which is largely identical with the previous reports of Kamel et al.30 on RA and DM. Among B cell LPD, 2 cases with polymorphic LPD were EBV-positive as is usually observed in cases of polymorphic PT-LPD.1 The positive rate of EBV in the B cell LPD with a monomorphous pattern of proliferation was 9.7%, which is rather close to that in NHL in the general population in Japan (7%).31 Whereas the EBV-positive rate in the present T cell LPD (76%) was much higher than in peripheral T cell (about 30%) and B cell lymphoma developing in the general population.32

In the current series, B cell LPD developed in four patients with RA who received MTX medication. EBV could not be detected in any of these patients. Therefore, the pathogenesis of LPD that developed in AID could not be explained only by the immunosuppressive state induced by MTX. Other factors such as a hyperimmune state might be causative of LPD in AID patients. Further study concerning this point is necessary.

Latent infection genes of EBV, especially LMP-1 and EBNA 2, show transforming activity in infected cells. These molecules, however, serve as targets for host cytotoxic T-lymphocytes (CTL). In the immunodeficient condition, lymphoid cells expressing these molecules can escape from host CTL, and thus might result in the development LPD. This might explain the increased risk of malignant lymphoma in immunodeficient individuals.

According to the expression pattern of EBV latent genes, EBV-associated malignancies are categorized into 3 groups; Lat I (EBNA-1⁺, LMP-1⁻, EBNA-2⁻) including Burkitt's lymphoma, Lat II (EBNA-1⁺, LMP-1⁺, EBNA-2⁻) including HL and nasopharyngeal carcinoma, and Lat III (EBNA-1⁺, LMP-1⁺, EBNA-2⁺) including LPD arising in immunocompromised patients. LPD in the present cases were LMP-1⁺ and EBNA-2⁻, and were thus categorized as Lat II,33 which differs from immunosuppressive LPD.

The 5-year overall survival rate in the present patients (33%) was rather more favorable than that in sporadic LPD (20.6%) and PT-LPD (26.7%), although the difference was not significant. LPD with B cell phenotype showed a more favorable 1-year survival rate (60%) than that with T cell phenotype (27%) and HL (33%) (p < 0.05). HL developing in RA patients who received MTX medication was reported to have a favorable prognosis.34 The current 2 cases of HL, LP and LD type, died within 1 year after the diagnosis. One of these cases presented with advanced disease and the other did not receive chemotherapy for LPD because the correct diagnosis was only possible just before death. Multivariate analysis showed that only the type of AID was an independent factor for the survival of patients, i.e., LPD in RA showed the most favorable prognosis.

In conclusion, LPD developed in RA patients with a longer interval between the onset of AID and the diagnosis of LPD. These patients showed the most favorable prognosis compared to LPD in other AID. Medication of RA with NSAID might work to suppress tumor growth, and thus might have contributed to these findings. EBV might play a role in the pathogenesis of T cell and polymorphic B cell LPD and HL in AID. LPD in AID is different from that in immunosuppressive conditions with respect to a lower EBV-positive rate and latency II pattern of EBV infection.

Acknowledgements

The authors thank Mr. Y. Kabutomori (Osaka Univ.) and Mr. Y. Tani (Dako Japan Co. ltd.) for their technical assistance. We also thank the following pathologists and physicians for providing clinical information: Drs. T. Daita, N. Miyogawa (Asahikawa Medical School), M. Kanda (Asahikawa Municipal Hosp.), T. Itoh (Kushiro Red Cross Hosp.), S. Kimura (Sapporo Medical School), H. Suzuki (National Sendai Hosp.), F. Yuda (Yamagata Municipal Hosp. Saiseikan), M. Nagata (Tsukuba Univ.), S. Itoyama, H. Takeuchi (Saitama Medical School), T. Suzuki (Tokyo Police Hosp.), K. Suda (Juntendo Univ.), R. Ariwa (Otsuka Metropolitan Hosp.), N. Nakamura (Yokohama Municipal Hosp.), H Sugiura (Kawasaki Municipal Ida Hosp.), I Okayasu (Kitasato Univ.), Y. Tsutsumi (Tokai Univ.), M. Naitoh, T. Hasegawa, I. Emura, M. Arakawa (Niigata Univ.), T. Nojima (Kanazawa Medical School), K. Suzuki (National Kanazawa Hosp.), M. Kuroda (Fujita Health Univ.), K. Doishita (Fukui Prefectural Hosp.), M. Maeda, T. Toyohashi (Toyohashi Municipal Hosp.), K. Kawahara, A. Kurata (National Osaka Hosp.), K. Kawano (Osaka Rosai Hosp.), M. Tsujimoto (Osaka Police Hosp.), A. Kanamaru, H. Miyazato (Kinki Univ.), T. Yamagami (National Osaka Minami Hosp.), Y. Uemura, S. Mori (Kansai Medical School), S. Ishiguro, H. Nakanishi (Center for Cancer and Cardiovascular Diseases, Osaka), K. Maeda (NTT West Hosp.), H. Ueki, T. Manabe (Kawasaki Medical Univ.), Y. Monobe (Kawasaki Hosp.), H. Sakamoto (Kagawa Medical School), I. Matsumoto (Matsuyama Red Cross Hosp.), S. Takeshita (Kyusyu Medical Center), Y. Iwata (Kyusyu Koseinenkin Hosp.), K. Irie (Saga Koseikann Hosp.), K. Takahara (Nagasaki Atomic Bomb Hosp.), and S. Yonemitsu, M. Yamaguchi (Saga Medical Univ.)